sm80s1-4405

Planet Ocean
J.L. Pelegrí and D. Vaqué (eds)

Variability of planktonic and epiphytic vibrios in a coastal environment affected by Ostreopsis blooms

Judit Bellés-Garulera 1, Magda Vila 1, Encarna Borrull 1, Pilar Riobó 2, José M. Franco 2,
Maria Montserrat Sala 1

1 Institut de Ciències del Mar-CSIC, Pg. Marítim de la Barceloneta 37-49, 08003 Barcelona, Catalunya, Spain.
E-mail: msala@icm.csic.es
2 Instituto de Investigaciones Marinas, CSIC, Eduardo Cabello, 6, CP36208, Vigo, Spain.

Summary: Vibrios include several pathogenic bacteria that occur in aquatic environments. The presence of Vibrio has been assessed in many ecosystems by culture-based techniques. However, little is known on the contribution of vibrios in the sea, especially in areas subject to harmful algal blooms. A preliminary study in Sant Andreu de Llavaneres beach (NW Mediterranean) showed the presence of some Vibrio species during a recurrent bloom of the harmful benthic dinoflagellate Ostreopsis cf. ovata. In order to establish the importance of vibrios in a coastal area of the NW Mediterranean and to study the association with the dinoflagellate, we conducted a sampling monitoring for one year to quantify the concentration of vibrios both in the water (free-living and attached to particles) and in the epiphytic community of macroalgae. The aims were 1) to evaluate the relative abundance of Vibrio in the epiphytic and in the planktonic bacterial community, 2) to assess the percentage of free-living and attached vibrios in the planktonic community, and 3) to determine whether the presence of vibrios is associated with the blooms of the toxic dinoflagellate Ostreopsis or with other environmental parameters. For this purpose, a CARD-FISH molecular probe was applied for the specific detection of bacteria belonging to the genus Vibrio. Cells were quantified and the abundance of both particles and bacteria attached to particles were assessed. The maximum Vibrio concentration (1.3x104 cells ml–1 and 1.4x106 cells g–1 FW, for planktonic and epiphytic samples, respectively) was detected in September. Free-living vibrios contributed 0.38±0.24% to the total free-living planktonic community and 1.12±0.28% to the epiphytic bacterial community. However, their contribution was particularly high in the planktonic community attached to particles (17.37±20.49%). Although in the planktonic community Vibrio was found preferentially free-living (82.63±20.01%), particles are a niche for vibrios, since in particles vibrios may represent up to 72% of the total attached bacterial community. Abundance of planktonic Vibrio was correlated with Ostreopsis concentration and it is likely that they play a role in the wound infections suffered by beach users during the bloom.

Keywords: Vibrio; bacteria; particles; Mediterranean; HAB; dinoflagellates.

Variabilidad de vibrios planctónicos y epifíticos en un ambiente costero afectado por proliferaciones de Ostreopsis

Resumen: El género Vibrio incluye a varias bacterias patogénicas que se encuentran en ecosistemas acuáticos. La presencia de Vibrio se ha estimado en muchos ecosistemas mediante técnicas basadas en cultivos. Sin embargo, se conoce poco sobre la contribución de vibrios en el mar, especialmente en áreas afectadas por proliferaciones algales nocivas. Un estudio preliminar en la playa de Sant Andreu de Llavaneres (Mediterráneo NO) mostró la presencia de algunas especies de Vibrio durante una proliferación recurrente del dinoflagelado béntico nocivo Ostreopsis cf. ovata. Para poder establecer la relevancia de los vibrios en un área costera del Mediterráneo NO y estudiar su asociación con el dinoflagelado, realizamos un muestreo de monitoreo durante un año para cuantificar la concentración de vibrios tanto en el agua (de vida libre y adheridos a partículas) y en la comunidad epifítica de macroalgas con los objetivos de 1) evaluar la abundancia relativa de Vibrio en la comunidad bacteriana tanto planctónica como epifítica, 2) estimar el porcentaje de vibrios de vida libre y adheridos a partículas en la comunidad bacteriana planctónica y 3) determinar si la presencia de vibrios está relacionada con las proliferaciones del dinoflagelado Ostreopsis o con otros parámetros ambientales. Para este propósito, se aplicó una sonda molecular de CARD-FISH para la detección específica de bacterias pertenecientes al género Vibrio. Se cuantificaron las células y también la abundancia de partículas y de las bacterias adheridas a estas partículas. La máxima concentración de Vibrio (1.3x104 cels ml–1 y 1.4x106 cels g–1 PF, para muestras planctónicas y epifíticas, respectivamente) fue detectada en Septiembre. Los vibrios de vida libre contribuyeron un 0.38±0.24% al total de la comunidad bacteriana de vida libre y un 1.12±0.28% a la comunidad bacteriana epifítica. Sin embargo, su contribución fue especialmente elevada en la comunidad bacteriana adherida a partículas (17.37±20.49%). Aunque en la comunidad planctónica Vibrio se encontraba preferentemente no adheridos a partículas (82.63±20.01%), las partículas constituyen un nicho para vibrios, ya que pueden llegar a representar hasta un 72% de la comunidad bacteriana adherida a partículas. La abundancia de Vibrio en el plancton se correlacionó con la concentración de Ostreopsis, y es posible que éstos jueguen un papel en las infecciones de heridas que sufren los bañistas durante las proliferaciones algales.

Palabras clave: Vibrio; bacterias; partículas; HAB; dinoflagelados.

Citation/Como citar este artículo: Bellés-Garulera J., Vila M., Borrull E., Riobó P., Franco J.M., Sala M.M. 2016. Variability of planktonic and epiphytic vibrios in a coastal environment affected by Ostreopsis blooms. Sci. Mar. 80S1: 97-106. doi: http://dx.doi.org/10.3989/scimar.04405.01A

Editor: D. Vaqué.

Received: January 18, 2016. Accepted: April 21, 2016. Published: September 30, 2016.

Copyright: © 2016 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Conclusion
Acknowledgements
References

INTRODUCTIONTop

Vibrio is a genus of heterotrophic bacteria that is widely spread in the ocean. It includes several potential pathogens such as V. cholerae, and bacteria associated with food-borne diseases such as V. alginolyticus, V. parahaemolyticus and V. vulnificus (Thompson et al. 2004Thompson J.R., Randa M.A., Marcelino L.A. et al. 2004. Diversity and dynamics of a north Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70: 4103-4110.), and with other infective syndromes such as otitis, pharyngitis and wound infections (De Paola et al. 1990De Paola A., Hopkins L.H., Peeler J.T., et al. 1990. Incidence of Vibrio parahaemolyticus in US coastal waters and oysters. Appl. Environ. Microbiol. 8: 2299-2302., Mizunoe et al. 2000Mizunoe Y., Wai S.N., Ishikawa T. et al. 2000. Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation. FEMS Microbiol. Lett. 186: 115-120.). Therefore, there has been considerable economic and health interest in determining their presence in coastal ecosystems.

Several studies have associated the presence of Vibrio with dinoflagellates (Mourino-Perez et al. 2003Mourino-Perez R.R., Worden A.Z., Azam F. 2003. Growth of Vibrio cholerae O1 in red tide waters off California. Appl. Environ. Microbiol. 69: 6923-6931., Eiler et al. 2006Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.). Ostreopsis is a genus of epiphytic and planktonic dinoflagellates that is found mainly attached to macroalgae but also free-living in seawater (Vila et al. 2001Vila M, Garcés E., Masó M. 2001. Potentially toxic epiphytic dinoflagellate assemblages on macroalgae in the NW Mediterranean. Aquat. Microb. Ecol. 26: 51-60.). It attaches to substrates by means of a rusty-brown mucilage that it secretes (Honsell et al. 2013Honsell G., Bonifacio A., De Bortoli M., et al. 2013. New insights on cytological and metabolic features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): A multidisciplinary approach. PLOS ONE, 8: e57291.). The genus is known for its ability to produce potent biotoxins known as palytoxins and analogues (Lenoir et al. 2004Lenoir S., Ten-Hage L., Turquet J. et al. 2004. First evidence of palytoxin analogues from an Ostreopsis mascarenensis (Dinophyceae) benthic bloom in Southwestern Indian Ocean. J. Phycol. 40: 1042-1051.) and is widespread in the Mediterranean (Mangialajo et al. 2011Mangialajo L., Ganzin N., Accoroni S. et al. 2011. Trends in Ostreopsis proliferation along the Northern Mediterranean coasts. Toxicon 57: 408-420. and references therein). At Sant Andreu de Llavaneres beach, NW Mediterranean, a recurrent massive bloom is found during summer, formed basically by Ostreopsis cf. ovata but also accompanied on some occasions by Ostreopsis cf. siamensis (Penna et al. 2005Penna A., Vila M., Fraga S. et al. 2005. Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western Mediterranean Sea based on morphology, toxicity and internal transcribed spacer 5.8s rDNA sequences. J. Phycol. 41: 212-225., Battocchi et al. 2010Battocchi C., Totti C., Vila M., et al. 2010. Monitoring of toxic microalga Ostreopsis (Dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR based assay combined with light microscopy method. Mar. Pollut. Bull. 60:1074-1084., Vila et al. 2012bVila M., Riobó P., Bravo I. et al. 2012b. A three-year time series of toxic Ostreopsis blooming in a NW Mediterranean coastal site: Preliminary results. In: Pagou P. and Hallegraeff G. (eds). Proceeding of the 14th International Conference on Harmful Algae. ISSHA and IOC of UNESCO, pp. 111-113.). These blooms have often been related to respiratory symptoms in beach users or persons taking a nearshore walk in Mediterranean countries since the late 1990s (see Vila et al. 2016Vila M., Abós-Herràndiz R., Isern-Fontanet J., et al. 2016. Establishing the link between Ostreopsis cf. ovata blooms and human health impacts using ecology and epidemiology. Sci. Mar. 80S1: 107-115.) and are thought to have been the cause of a massive mortality of benthic invertebrates in Llavaneres in August 1998 (Vila et al. 2008Vila M., Masó M., Sampedro N. et al. 2008. The genus Ostreopsis in recreational waters of the Catalan Coast and Balearic Islands (NW Mediterranean Sea): is this the origin of human respiratory difficulties? In: Proceedings of the 12 International Conference on Harmful Algae, pp. 334-336.). The mortality of benthic invertebrates and fishes (Simoni et al. 2004Simoni F., di Paolo C., Gori L. et al. 2004. Further investigation on blooms of Ostreopsis ovata, Coolia monotis, Prorocentrum lima on the macroalgae of artificial and natural reefs in the Northern Tyrrhenian Sea. Harmful Algae News 26: 5-7., Shears and Ross 2009Shears N.T., Ross P.M. 2009. Blooms of benthic dinoflagellates of the genus Ostreopsis; an increasing and ecologically important phenomenon on temperate reefs in New Zealand and worldwide. Harmful Algae 8: 916-925) has been documented at other locations, as have other noxious effects, such as human food-borne intoxications (Tubaro et al. 2011Tubaro A., Durando P., Del Favero G. et al. 2011. Case definitions for human poisonings postulated to palytoxins exposure. Toxicon 57: 478-495.).

The bacterial community associated with harmful algal blooms (HABs) has been studied due to its possible role in the toxicity of dinoflagellate blooms (Gallacher et al. 1997Gallacher S., Flynn K.J., Franco J.M. et al. 1997. Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp (Dinophyta) in culture. Appl. Environ. Microbiol. 63: 239-245., Lu et al. 2000Lu Y. H., Chai T.J., Hwang D.F. 2000. Isolation of bacteria from toxic dinoflagellate Alexandrium minutum and their effects on algae toxicity. J. Nat. Toxins 9: 409-417., Sala et al. 2005Sala M.M., Balagué V., Pedrós-Alio C. et al. 2005. Phylogenetic and functional diversity of bacterioplankton during Alexandrium spp. blooms. FEMS Microbiol. Ecol. 54: 257-267., Kodama et al. 2006Kodama M., Doucette G.J., Green D.H. 2006. Relationships between bacteria and harmful algae. In: Granelli E. and Turner J.T. (eds) Ecology of Harmful Algae, Springer, pp: 243-255., Barlaan et al. 2007Barlaan E.A., Furukawa S., Kazuisha T. 2007. Detection of bacteria associated with harmful algal blooms from coastal and microcosm environments using electronic microarrays. Environ. Microbiol. 9: 690-702.). This contribution might be due to bacterial lysis of algal cells or the active release of bacterial toxins (Lenes et al. 2013Lenes J.M., Walsh J.J., Barrow B.P. 2013. Simulating cell death in the termination of Karenia brevis blooms: implications for predicting aerosol toxicity vectors to humans. Mar. Ecol. Prog. Ser. 493: 71-81.).

Studies of the microbial community associated with Ostreopsis started some decades ago (Tosteson et al. 1989Tosteson T.R., Ballantine D.L., Tosteson C.G. et al. 1989. Associated bacterial-flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus-toxicus. Appl. Environ. Microbiol. 55: 137-141., Ashton et al. 2003Ashton M., Rosado W., Govind N.S. et al. 2003. Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis. Toxicon 42: 419-424., among others) and were conducted with cultures. They highlighted the need of symbiotic bacteria for the growth of Ostreopsis (Ashton et al. 2003Ashton M., Rosado W., Govind N.S. et al. 2003. Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis. Toxicon 42: 419-424.); the bacterial community in Ostreopsis cultures was dominated by genus Vibrio or Alteromonas, in the class of γ-proteobacteria and by the complex Cytophaga-Flavobacter-Bacteroidetes (Tosteson et al. 1989Tosteson T.R., Ballantine D.L., Tosteson C.G. et al. 1989. Associated bacterial-flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus-toxicus. Appl. Environ. Microbiol. 55: 137-141., Ashton et al. 2003Ashton M., Rosado W., Govind N.S. et al. 2003. Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis. Toxicon 42: 419-424., Pérez-Guzman et al. 2008Pérez-Guzman L., Pérez-Matos A.E., Rosado W. et al. 2008. Bacteria associated with toxic clonal cultures of the dinoflagellate Ostreopsis lenticularis. Mar. Biotech. 10: 492-496.).

A preliminary study aimed at characterizing the bacterial community associated with field Ostreopsis blooms in Llavaneres beach (Borrull 2011Borrull E. 2011. Diversidad y actividad de la comunidad microbiana asociada a proliferaciones de fitobentos tóxico. Master thesis. Universitat de Barcelona.) detected the presence of three OTUS of the genus Vibrio in epiphytic samples during the bloom of summer 2010: Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio tubiashi. However, the technique used (DGGE) at that time could not provide information on their abundance. Therefore, the present study aims at determining the factors that drive the abundance of vibrios in the planktonic and epiphytic community and their potential contribution to the toxicity of the bloom.

Most studies on the occurrence of Vibrio in natural environments have been based on culture-dependent techniques, but some Vibrio may be unable to grow on conventional media, so molecular techniques are more appropriate. Although several studies have focused on the detection of the genus or certain species, data on the abundance of Vibrio in seawater are scarce and restricted to the Baltic and Skagerrak Seas (Eiler et al. 2006Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.), the Arabian Sea (Gallacher et al. 1997Gallacher S., Flynn K.J., Franco J.M. et al. 1997. Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp (Dinophyta) in culture. Appl. Environ. Microbiol. 63: 239-245.) and the North Sea (Oberbeckmann et al. 2012Oberbeckmann S., Fuchs B.M., Meiners M. et al. 2012. Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea. Microb. Ecol. 63: 543-551.). Although Vibrio seems to be an important pathogenic agent in the NW Mediterranean (e.g. Canigral et al. 2010Canigral I., Moreno Y., Alonso J.L., et al. 2010. Detection of Vibrio vulnificus in seafood, seawater and wastewater samples from a Mediterranean coastal area. Microbiol. Res. 165: 657-664., Lopez-Joven et al. 2015Lopez-Joven C., de Blas I., Furones M.D. et al. 2015. Prevalences of pathogenic and non-pathogenic Vibrio parahaemolyticus in mollusks from the Spanish Mediterranean Coast. Front. Microbiol. 6: 736.), to the best of our knowledge no information on abundance of planktonic Vibrio (using culture independent methods) is available for Mediterranean waters.

In the present study we carried out a sampling of both the planktonic and the epiphytic bacterial community in Sant Andreu de Llavaneres beach (NW Mediterranean), which is regularly affected by blooms of Ostreopsis in the summer months. Borrull (2011)Borrull E. 2011. Diversidad y actividad de la comunidad microbiana asociada a proliferaciones de fitobentos tóxico. Master thesis. Universitat de Barcelona. reported on the importance of epiphytic Vibrio in the summer months, but did not assess its abundance. We therefore hypothesize a higher relevance of Vibrio in the epiphytic than in the planktonic communities, and also that Vibrio abundance is associated with Ostreopsis blooms.

Traditionally, vibrios have been generally thought to be associated with animals, probably because they were investigated only in intoxicated tissues (Thompson et al. 2004Thompson J.R., Randa M.A., Marcelino L.A. et al. 2004. Diversity and dynamics of a north Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70: 4103-4110.), and they have been found attached to several marine organisms (see Takemura et al. 2014Takemura A.E., Chien D.M., Polz M.E. 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front. Microbiol. 5: 38. for a review) and also phytoplankton (e.g. Tamplin et al. 1990Tamplin M.L., Gauzens M.L., Huq A. et al. 1990. Attachment of Vibrio-cholerae serogroup-O1 to zooplankton and phytoplankton of Bangladesh waters. Appl. Environ. Microb. 56: 1977-1980., Neogi et al. 2012Neogi S.B., Islam M.S., Nair G.B. et al. 2012. Occurrence and distribution of plankton-associated and free-living toxigenic Vibrio cholerae in a tropical estuary of a cholera endemic zone. Wet. Ecol. Manag. 20: 271-285.). However, in some recent studies both free-living lifestyles or communities associated with aggregates have been reported for Vibrio (Lyons et al. 2007Lyons M.M., Lau Y.T., Carden W.E. et al. 2007. Characteristics of marine aggregates in shallow-water ecosystems: Implications for disease ecology. Ecohealth 4: 406-420., Froelich et al. 2013Froelich B., Ayrapetyan M., Oliver J.D. 2013. Integration of Vibrio vulnificus into marine aggregates and its subsequent uptake by Crassostrea virginica oysters. Appl. Environ. Microbiol. 79: 1454-1458., Szabo et al. 2013Szabo G., Preheim S.P., Kauffman K.M. et al. 2013. Reproducibility of Vibrionaceae population structure in coastal bacterioplankton. ISME J. 7: 509-519.). A second hypothesis of our study is therefore that Vibrio is found more in particles than free-living in plankton.

Due to the direct and indirect effects on human health, Vibrio can impact the local economy by affecting tourism, fisheries and aquaculture. Therefore, the prevalence of Vibrio spp. in coastal environments is of concern and the factors that regulate its dynamics need to be elucidated.

MATERIALS AND METHODSTop

Study area and sample collection

The study was carried out in Sant Andreu de Llavaneres beach (41°33.130’N, 2°29.540’E) in the NW Mediterranean from January to December 2010. The area is a fossil rocky beach that is highly colonized by different genera of macroalgae of the genera Corallina, Jania, Halopteris, Dyctyota and Padina, among others. Sampling was done monthly in winter and spring and the frequency was increased during the summer and autumn months, when Ostreopsis was detected. Both seawater and macrophyte fragments were collected in each sampling.

Environmental parameters in the water

Temperature and salinity were measured with a WTW Model LF 197 microprocessor conductivity meter. Chlorophyll-a determination followed the method in Yentsch and Menzel (1963)Yentsch C.S., Menzel D.W. 1963. A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res. 10: 221-231.. Briefly, 60 ml of surface water samples were filtered through GF/F glass fibre filters and frozen at –20°C until analysis. Samples were extracted in 6 ml of 90% acetone for 24 h at 4°C, and chlorophyll-a was measured with a Turner Designs fluorimeter. For inorganic nutrient analyses, 60 ml water samples were taken and frozen (–20°C). Analyses of dissolved inorganic nutrients (NO3, NO2, NH4, PO4 and SiO4) were performed as described by Grasshoff et al. (1983)Grasshoff H., Ehrhardt M., Kremling K. 1983. Methods of Seawater Analysis. Verlag Chemie, Germany. with a Seal Analytical AA3 continuous flow analyser (Bran+Luebbe).

Bacterial and particle abundance

Samples from both seawater and macroalgae were collected. Fragments of macroalgae (generally Corallina) of 2-7 g were placed in plastic tubes and filled up to 50 ml with in situ seawater filtered through 0.2 µm pore size filters. Water samples were collected using polyethylene acid-rinsed bottles. All samples were carried to the lab in the dark for further processing within two hours of collection. Once in the lab, the macroalgae solution was diluted with in situ filtered (0.2 µm) seawater up to 200 ml. The bottle was also vigorously shaken with a vortex mixer to detach organisms from the mucilage. The dilution was finally filtered through a mesh (140-200 µm) and this final solution was used for further processing. Both macroalgae and seawater samples were processed to assess particle size and density as well as abundance of both total bacteria and specific groups.

Bacterial and particle counts were assessed microscopically after staining with DAPI (4’,6-diamino-2-fenilindol) according to Porter and Feig (1980)Porter K.G., Feig F.Y. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948.. Briefly, after fixing samples with glutaraldehyde (3.6% final concentration), samples were filtered through a black Millipore filter with 0.2 µm pore size. The filters were stained with DAPI (final conc. 1 mg ml–1) and observed under an epifluorescence microscope Olympus BX61. Discrete fields were counted for bacterial abundance, whereas for particles the filters were scanned with several transects in which the length and width of the particles were also measured. Particle size was calculated by assuming a square size.

Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was used for the analysis of the abundance of single bacterial groups. We followed the protocol of Pernthaler et al. 2002Pernthaler A., Pernthaler J., Amann R. 2002. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68: 3094-3101.), which is similar to that in Alonso-Sáez et al. (2007)Alonso-Sáez L., Balagué V., Sà E.L., et al. 2007. Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH. FEMS. Microbiol. Ecol. 60: 98-112. and Ruiz-González et al. (2012)Ruiz-González C., Lefort T., Galí M. et al. 2012 Seasonal patterns in the sunlight sensitivity of bacterioplankton from Mediterranean surface coastal waters. FEMS Microbiol. Ecol. 79: 661-674. We used two horseradish peroxidase-labelled probes to identify bacterial groups in the samples: GAM42, which targets most of the γ-proteobacteria (55% formamide; Manz et al. 1992Manz W., Amann R., Ludwig W., et al. 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria - problems and solutions. Syst. Appl. Microbiol.15: 593-600.), and VIB572a, which targets most of Vibrio (50% formamide; Huggett et al. 2008Huggett M.J., Crocetti G.R., Kjelleberg S. 2008. Recruitment of the sea urchin Heliocidaris erythrogramma and the distribution and abundance of inducing bacteria in the field. Aquat. Microb. Ecol. 53: 161-171.). The probe VIB572a covers 15 different Vibrio strains that include V. alginolyticus, V. parahemolyticus, V. vulnificus, V. cholera, and also four Photobacterium strains (Huggett et al. 2008Huggett M.J., Crocetti G.R., Kjelleberg S. 2008. Recruitment of the sea urchin Heliocidaris erythrogramma and the distribution and abundance of inducing bacteria in the field. Aquat. Microb. Ecol. 53: 161-171., Fig. 1). Briefly, 4.5 ml of seawater or of the epiphyte solution were fixed overnight with paraformaldehyde (1%) at 4°C. Samples were gently filtered on 0.2-µm Millipore polycarbonate filters. Filters were permeabilized with lysozyme (37°C, 1 h) and achromopeptidase (37°C, 30 min) before hybridization. Hybridizations were carried out overnight at 35°C with a percentage of formamide of 50% and 55% for GAM42a and VIB572a, respectively. The Beta42a (Manz et al. 1992Manz W., Amann R., Ludwig W., et al. 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria - problems and solutions. Syst. Appl. Microbiol.15: 593-600.) antisense probe was used as a negative control. For amplification, we used tyramide labelled with Alexa 488. Counterstaining of CARD-FISH preparations was done with DAPI (final concentration 1 mg ml–1). DAPI and FISH-stained cells were counted. For γ-proteobacteria, between 500 and 1,000 positive cells were counted manually in a minimum of 30 fields. For Vibrio, due to the lower concentration, between 1 and 4 transects of the filters were scanned.

Phytoplankton abundance

Seawater samples for enumeration of planktonic Ostreopsis were fixed with lugol. An aliquot of 10-50 ml was placed in a counting chamber for 24 h, and for enumeration of the phytoplankton cells an area of the sample was scanned at 63-400× depending on cell density using a Leika-Leitz DM-II inverted microscope. For the abundance of epiphytic Ostreopsis, fragments of macroalgae (generally Corallina) of 10-20 g were placed in plastic bottles and filled up to 120 ml with in situ GF/F filtered seawater. They were shaken vigorously for 1 min to detach organisms from the macroalgae, and the solution was filtered through a mesh (140 µm) in order to separate the macroalgae and the bigger organisms. The samples were then fixed with Lugol’s solution. An aliquot of 1-10 ml was placed in a Sedgwick-Rafter or Utermöhl chamber and Ostreopsis cells were counted as indicated above. The abundance of epiphytic phytoplankton was expressed as cell per gram of fresh weight of macroalgae.

Statistical analysis

Pearson’s correlations were performed with STATISTICA software, version 8.0 (StaSoft).

RESULTSTop

Environmental parameters of the water

The thermohaline characteristics of the waters at Sant Andreu de Llavaneres in 2010 differed over the year (Fig. 1), with minimum temperatures in winter (13.0°C) and maximum temperatures in summer (25.7°C). Salinity ranged between 37.1 and 37.8, with no clear seasonal pattern. Chlorophyll-a concentrations ranged from 0.3 µg l–1 in July to 8.1 µg l–1 in November (data not shown).

sm4405fig1.jpg

Full size image

Fig. 1. – Temperature and salinity in the water of Sant Andreu de Llavaneres Beach in 2010.

Abundance of Ostreopsis

Abundance of Ostreopsis cells (both epiphytic and planktonic) was below the detection limit during the first part of the year (Fig. 2) and started increasing in June-July to achieve a first peak in August and a second peak in September-October. A huge peak of Ostreopsis that turned the water brown-red was found in September (9.9×106 cells l–1), when epiphytic Vibrio also achieved a peak of 1.4×106 cells g–1 FW.

sm4405fig2.jpg

Full size image

Fig. 2.Ostreopsis sp. abundance in the plankton and in the epiphytic community of the macrophytes in 2010.

Total bacterial and Vibrio abundance

Both bacterial and Vibrio abundance were assessed by epifluorescence microscopy (see photos in Fig. 3). Planktonic bacterial concentrations achieved their highest values in summer and autumn, with a maximum in September (1.5×106 cells ml–1; Fig. 4A). Epiphytic bacterial concentrations also showed higher values between June and October and peaked in August (5.3×108 cells g–1 FW), but showed higher variability (Fig. 4B).

sm4405fig3.jpg

Full size image

Fig. 3. – Epifluorescence microscopy of planktonic bacteria stained with DAPI (A), hybridized with the Vibrio CARD-FISH probe after amplification with tyramide-Alexa488 (B) and stained with DAPI and attached to an Ostreopsis cell (C).

sm4405fig4.jpg

Full size image

Fig. 4. – Total bacterial and Vibrio abundance, and Vibrio percentage of total cells in the plankton (free-living + attached) (A) and epiphytic community (B) in 2010.

Planktonic vibrios showed a similar trend to that of total planktonic bacterial concentration, with higher concentrations in late summer and autumn, and a peak in September of 1.3×104 cells ml–1. However, the range of percentage contribution to total bacterial concentration was low and varied between 0.11 and 0.86% (mean 0.38%), with the highest values in summer and winter (Fig. 4A).

The trend of epiphytic vibrios was similar to that of the total epiphytic bacteria. Concentrations varied between 7.1×104 and 1.1×107 cells g–1 FW, and the highest peaks were observed in July and August. The percentage contribution of Vibrio to total epiphytic bacteria was also low but higher than in the plankton (mean of 1.13%), with peaks in July of 4.3% (Fig. 4B).

Bacteria attached to particles

Seawater contained between 11 and 417 particles ml–1, with higher concentrations between August and September (Fig. 5). A similar pattern was observed for the number of bacteria attached to particles, which varied between 380 and 8285 cells ml–1, which corresponded to between 0.14 and 4.0% of total bacteria, with a mean of 0.8% (data not shown).

sm4405fig5.jpg

Full size image

Fig. 5. – Particle abundance and bacteria attached to particles in 2010.

Vibrio cells appeared to be mostly free-living (353-11851 cells ml–1), with higher values in summer and autumn (Fig. 6) representing a mean of 82.10±20.01% of the total concentration of planktonic vibrios. Concentration of vibrios attached to particles was low (42-3451 cells ml–1), and the highest values were achieved towards the end of the year, in autumn. In fact, from October to December the abundance of free-living vibrios and vibrios attached to particles was quite similar. These differences in the period of dominance of the free vs attached Vibrio population deliver large differences in the percentage contribution of Vibrio to the attached bacterial community, which varied from 1.5% in the summer months to around 12%-25% in spring, and up to 74% in December.

sm4405fig6.jpg

Full size image

Fig. 6. – Concentration of planktonic free-living and attached Vibrio, and percentage contribution of vibrios to the attached bacterial community.

Mean percentage contribution of vibrios to the total bacterial community varied among the communities (Fig. 7). For the planktonic community, free-living vibrios accounted for a mean of 0.38% (range 0.11-0.86%) of total free-living bacteria. The epiphytic community on the macrophyte contained a larger percentage of vibrios (mean 1.13%; range: 0.07-4.25%). The highest contribution of Vibrio was found in the bacterial community attached to particles in the plankton (mean 17.32%; range:1.47-73.90%).

sm4405fig7.jpg

Full size image

Fig. 7. – Percentage contribution of Vibrio to total planktonic (free-living or attached to particles) and epiphytic bacterial communities.

Correlations among parameters

Pearson’s correlation coefficients between bacterial and particle parameters and several physico-chemical and biological parameters are shown in Table 1. Abundance of both total and free-living planktonic vibrios correlated positively with Ostreopsis concentration. Ostreopsis also correlated positively with the number of particles in the water. Abundance of total epyphitic and planktonic vibrios, and percentage of epiphytic vibrios of the total bacterial community showed a significant positive correlation with temperature. Percentage of γ-proteobacteria, the taxonomical group to which Vibrio belongs, showed a significant positive correlation with ammonia. Abundance of planktonic bacteria showed a significant positive correlation with both temperature and Ostreopsis concentration, and a significant negative correlation with total inorganic nutrient concentration.

Table 1. – Pearson correlation coefficients between epiphytic (E) and planktonic (P) concentration of bacteria, vibrios and particles and selected abiotic and biotic variables at Sant Andreu de Llavaneres in 2010; n=13-17. Significant correlations (p<0.05) are indicated in bold. TIN, total inorganic nitrogencaption

Temp Salinity Chl a NO3+NO2 NH4 TIN Phosphate Silicate Ostreopsis
Bacteria E 0.09 –0.10 0.23 0.14 0.12 0.10 0.08 –0.10 0.38
P 0.64 –0,43 0.11 –0.43 –0.24 –0.48 0.11 –0.37 0.57
Vibrio total E 0.62 –0.37 –0.17 –0.34 0.15 –0.20 –0.18 –0.38 0.36
P 0.66 0.23 0.15 –0.25 –0.21 –0.33 0.02 –0.03 0.47
Vibrio free P 0.64 –0.24 –0.00 –0.32 –0.13 –0.33 –0.55 –0.09 0.52
Vibrio attached P –0.03 –0.24 0.42 –0.09 –0.18 –0.07 0.21 0.01 –0.06
% γ-Proteobacteria E 0.29 –0.25 –0.50 –0.04 –0.17 –0.07 –0.19 0.08 –0.28
P 0.09 –0.12 –0.20 0.05 0.57 0.14 0.14 –0.31 –0.25
%Vibrio E 0.70 –0.44 –0.39 –0.55 0.11 –0.34 –0.33 –0.44 0.22
P 0.52 –0.06 0.15 –0.11 –0.17 –0.18 0.01 0.19 0.32
%Vibrio attached P –0.45 –0.18 0.35 0.24 –0.06 0.13 0.22 0.15 –0.37
Particles ml–1 P 0.29 –0.19 0.18 –0.10 0.06 –0.09 0.06 –0.33 0.54

DISCUSSIONTop

Epiphytic and planktonic blooms of the dinoflagellate Ostreopsis cf. ovata occur recurrently at several localities of the Mediterranean Sea (Mangialajo et al. 2011Mangialajo L., Ganzin N., Accoroni S. et al. 2011. Trends in Ostreopsis proliferation along the Northern Mediterranean coasts. Toxicon 57: 408-420.). The relationship of these blooms with bacteria has rarely been analysed. Surprisingly, bacterial concentrations in Llavaneres were very similar to those found during the year in Blanes Bay, a coastal location on the Catalan coast (Alonso-Sáez et al. 2008Alonso-Sáez L., Vázquez-Domínguez E., Pinhassi J. et al. 2008. Factors controlling the year-round variability in carbon flux through bacteria in a coastal marine system. Ecosystems 11: 397-409.), despite the fact that the sampling station at Llavaneres is shallower (50 cm depth) and close to anthropogenic influences, and has higher chlorophyll concentrations and occasional mucilage loads coinciding with the Ostreopsis blooms.

Is the epiphytic community more enriched in vibrios than the planktonic bacterial community?

Our study shows a temporal variation in the abundance of Vibrio spp. in a coastal marine area of the NW Mediterranean in both the epiphytic and planktonic communities, with higher concentrations in the warm period. With the specific CARD-FISH probe, we have provided the first values of the concentration of vibrios in Mediterranean coastal waters. However, we are aware that the coverage of our probe may be incomplete (see details in Huggett et al. 2008Huggett M.J., Crocetti G.R., Kjelleberg S. 2008. Recruitment of the sea urchin Heliocidaris erythrogramma and the distribution and abundance of inducing bacteria in the field. Aquat. Microb. Ecol. 53: 161-171.) and is not specific enough to monitor the different Vibrio populations that may appear at different stages of the year cycle (Thompson et al. 2005Thompson J.R., Pacocha S., Pharino C. et al. 2005. Genotypic diversity within natural coastal bacterioplankton population. Science 307: 1311-1313.) The percentages of total Vibrio in the plankton detected at Sant Andreu de Llavaneres (mean 0.38%, maximum 0.9% in August) are slightly lower than the values detected recently in the North Sea with CARD-FISH (2%; Oberbeckmann et al. 2012Oberbeckmann S., Fuchs B.M., Meiners M. et al. 2012. Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea. Microb. Ecol. 63: 543-551.), higher than those in the Baltic Sea detected with qPCR (0.002%-0.015%; Eiler et al. 2006Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.), but in the same range as those in the Arabian Sea (0.2%-1.3%; Asplund et al. 2011Asplund M.E., Rehnstam-Holm A.S., Atnur V., et al. 2011. Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area. Environ. Microbiol. 13: 2738-2751.).

The mean contribution of epiphytic vibrios was three times that of planktonic bacteria (mean 1.1%, with a peak of 4.2% of total epiphytic bacterial community). Presence of Vibrio on marine macroalgae has been well documented and Vibrio is the dominant culturable bacteria in several red algae (Takemura et al. 2014Takemura A.E., Chien D.M., Polz M.E. 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front. Microbiol. 5: 38. and references therein). Apparently, macroalgae can serve as a refuge for vibrios, especially non-native macroalgal species (Gonzalez et al. 2014Gonzalez D.J., Gonzalez R.A., Froelich B.A. et al. 2014. Non-native macroalga may increase concentrations of Vibrio bacteria on intertidal mudflats. Mar. Ecol. Prog. Ser. 505: 29-36.). Together with temperature, trophic resource availability is one of the most important controls of Vibrio abundance (Oberbeckman et al. 2012Oberbeckmann S., Fuchs B.M., Meiners M. et al. 2012. Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea. Microb. Ecol. 63: 543-551., Cavallo and Stabili 2004Cavallo R.A., Stabili L. 2004. Culturable vibrios biodiversity in the Northern Ionian Sea (Italian coasts). Sci. Mar. 68: 23-29.). Increased nutrient loads at this coastal station might have enhanced the growth of γ-proteobacteria and also of Vibrio, although a correlation between the latter and inorganic nutrients could not be found.

This study contributes to the knowledge that vibrios have a small representation in the planktonic bacterial community. However, their contribution in the epiphytic community is three times higher. Although numbers of planktonic vibrios are low, their biomass can be up to 100 times higher than that of SAR11, and they are known to play an important role in the ecosystem through biodegradation, nutrient regeneration and biogeochemical cycling (for example, as chitin degraders) (Takemura et al. 2014Takemura A.E., Chien D.M., Polz M.E. 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front. Microbiol. 5: 38.).

Are vibrios found preferentially attached to particles?

Vibrios have been detected on a large variety of biological surfaces, especially animals (Thompson et al. 2004Thompson J.R., Randa M.A., Marcelino L.A. et al. 2004. Diversity and dynamics of a north Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70: 4103-4110., Baffone et al. 2006Baffone W., Tarsi R., Pane L. et al. 2006. Detection of free-living and plankton-bound vibrios in coastal waters of the Adriatic Sea (Italy) and study of their pathogenicity-associated properties. Environ. Microbiol. 8: 1299-1305., Main et al. 2015Main C.R., Salvitti L.R., Whereat E.B. et al. 2015. Community-level and species-specific associations between phytoplankton and particle-associated Vibrio species in Delaware’s inland bays. Appl. Environ. Microbiol. 81: 5703-5713.), and are also associated with various types of organic particles of non-animal origin (Lyons et al. 2007Lyons M.M., Lau Y.T., Carden W.E. et al. 2007. Characteristics of marine aggregates in shallow-water ecosystems: Implications for disease ecology. Ecohealth 4: 406-420., Froelich et al. 2013Froelich B., Ayrapetyan M., Oliver J.D. 2013. Integration of Vibrio vulnificus into marine aggregates and its subsequent uptake by Crassostrea virginica oysters. Appl. Environ. Microbiol. 79: 1454-1458.). Recently, there has been evidence that vibrios can remain free-living (Mourino-Perez et al. 2003Mourino-Perez R.R., Worden A.Z., Azam F. 2003. Growth of Vibrio cholerae O1 in red tide waters off California. Appl. Environ. Microbiol. 69: 6923-6931., Worden et al. 2006Worden A.Z., Seidel M., Smriga S. et al. 2006. Trophic regulation of Vibrio cholerae in coastal marine waters. Environ. Microbiol. 8: 21-29., Eiler et al. 2006Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.), although little is known on the factors determining whether they remain free-living versus particle-attached (Takemura et al. 2014Takemura A.E., Chien D.M., Polz M.E. 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front. Microbiol. 5: 38.). Whereas attachment to biofilms may provide a refuge from protozoan predation, attachment to particles may increase their susceptibility to being grazed by macrofauna but also increase their dispersal.

In the summer months, abundance of particles in the water showed a four-fold increase. In this study we frequently observed fragments of Ostreopsis thecae, and also mucilage associated with the dinoflagellates, which contributed to the pool of particles. Indeed, concentration of particles correlated with abundance of Ostreopsis in the water. The mucilage is rich in carbohydrates (Mestre pers. comm.) and the thecae in cellulose, both good sources of organic carbon for bacterial growth, which may favour particle colonization.

The contribution of plankton-attached vibrios to the total attached bacterial community was much higher (mean 4.0%, maximum 73.9%) than that of free-living vibrios to total free-living bacteria (mean 0.8%, maximum 17.37%). Particles concentrate more than half of the Vibrio population in the water, especially in autumn coinciding with the decaying Ostreopsis bloom. It is noteworthy that the lowest percentages of attached Vibrio are found in summer, when the concentration of attached bacteria is higher. Our data on the high percentage of free-living planktonic Vibrio (mean 83.8%, range 26.1%-99.5%) contribute to recent knowledge that, contrary to early studies, places Vibrio as a predominantly free-living bacteria, with comparable percentages (73-89%) to those in the Baltic Sea (Eiler et al. 2006Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.).

Is abundance of vibrios linked to Ostreopsis concentration?

Although studies have shown a correlation between vibrios and chlorophyll-a concentration (e.g. Asplund et al. 2011Asplund M.E., Rehnstam-Holm A.S., Atnur V., et al. 2011. Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area. Environ. Microbiol. 13: 2738-2751.), we found no relation between them in our study. Quantitative and qualitative differences in phytoplankton species composition may lead to pronounced differences in bacterioplankton species composition (Pinhassi et al. 2004Pinhassi J., Sala M.M., Havskum H. et al. 2004. Changes in bacterioplankton composition under different phytoplankton regimes. Appl. Environ. Microbiol. 70: 6753-6766.). In particular, the relationship between vibrios and specific groups of phytoplankton is controversial, and some authors suggest a preferential association with dinoflagellates (Eiler et al. 2006Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.), while others suggest a minor link between the two groups (Main et al. 2015Main C.R., Salvitti L.R., Whereat E.B. et al. 2015. Community-level and species-specific associations between phytoplankton and particle-associated Vibrio species in Delaware’s inland bays. Appl. Environ. Microbiol. 81: 5703-5713.). Our data show a positive relation between the abundances of vibrios and of the dinoflagellate Ostreopsis at Llavaneres. Such a relationship between the two genera was already established in early studies, when Vibrio sp. was isolated as an important bacteria in Ostreopsis cultures (Tosteson et al. 1989Tosteson T.R., Ballantine D.L., Tosteson C.G. et al. 1989. Associated bacterial-flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus-toxicus. Appl. Environ. Microbiol. 55: 137-141.).

Bacterial composition during HABs is subject to study due to the possible contribution to the toxicity of the blooms (Groben et al. 2000Groben R., Doucette G.J., Kopp M., et al. 2000. 16S rRNA targeted probes for the identification of bacterial strains isolated from cultures of the toxic dinoflagellate Alexandrium tamarense. Microb. Ecol. 39: 186-196.); for example, toxigenic bacteria may contribute half the algal-associated PSP toxin levels in Alexandrium cultures, provided that there is physical contact with the alga (Doucette et al. 1998Doucette G.J., Kodama G., Franca S. 1998. Bacterial interactions with harmful algal bloom species: Bloom ecology, toxigenesis, and cytology. In: Andersen D.M., Cembella A.D., Hallegraeff G.M. (eds) The Physiological Ecology of Harmful Algal Blooms, NATO/ASI Series. Springer Verlag, Heidelberg, pp. 619-646.). At Sant Andreu de Llavaneres, coinciding with Ostreopsis blooms, beach users often suffer from skin irritation and wound infections. These symptoms have often been attributed to some Vibrio species, with severe cases of V. vulnificus in the Baltic Sea (Ruppert et al. 2004Ruppert J., Panzig B., Guertler L. et al. 2004. Two cases of severe sepsis due to Vibrio vulnificus wound infection acquired in the Baltic Sea. Eur. J. Clin. Microbiol. Infect. Dis. 23: 912-915.) and of V. alginolyticus in the North Atlantic (Shets et al. 2006Shets F.M., van den Berg H.H., Demeulmeester A.A. et al. 2006. Vibrio alginolyticus infections in the Netherlands after swimming in the North Sea. Euro Surveill. 11: 3077., Reilly et al. 2011Reilly G.D., Reilly C.A., Smith E.G. et al. 2011. Vibrio alginolyticus-associated wound infection acquired in British waters, Guernsey. Euro Surveill., 16: 1994). In order to establish a connection between wound infections and the presence of pathogenic vibrios during Ostreopsis blooms, further research is being conducted to assess toxin profiles and identify the Vibrio species present at Llavaneres.

Relationships between Vibrio and environmental factors

Temperature is an important factor for the growth of Vibrio (Thompson et al. 2004Thompson J.R., Randa M.A., Marcelino L.A. et al. 2004. Diversity and dynamics of a north Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70: 4103-4110., Vezzulli et al. 2013Vezzulli, L., Colwell R.R., Pruzzo C. 2013. Ocean warming and spread of pathogenic vibrios in the aquatic environment. Microb. Ecol. 65: 817-825.) and it was the most important environmental parameter correlating positively with the abundance of both epiphytic and planktonic vibrios, both total and free-living concentrations, and the percentage contribution of vibrios to the community. Indeed, long-term studies have provided evidence of a significant positive relationship between sea surface temperature and Vibrio occurrence (Vezzulli et al. 2012Vezzulli L., Brettar I., Pezzati E. et al. 2012. Long-term effects of ocean warming on the prokaryotic community: evidence from the vibrios. ISME J. 6: 21-30.). In the NW Mediterranean, Vibrio infection together with temperature might have contributed to mass mortality events of benthic invertebrates (Vezzulli et al. 2010Vezzulli L., Previati M., Pruzzo C. et al. 2010. Vibrio infections triggering mass mortality events in a warming Mediterranean Sea. Environ. Microbiol. 12: 2007-2019.). On the Adriatic Sea coast, Vibrio expressing pathogenicity-associated properties were found mainly in the warmer months (Baffone et al. 2006Baffone W., Tarsi R., Pane L. et al. 2006. Detection of free-living and plankton-bound vibrios in coastal waters of the Adriatic Sea (Italy) and study of their pathogenicity-associated properties. Environ. Microbiol. 8: 1299-1305.).

Llavaneres is a very shallow beach and might be influenced by occasional seepage water from surrounding land. Salinity varies without a clear pattern probably due to these terrestrial influences of seepage water. Some studies have found positive correlations between vibrios and low salinities (Oberbeckmann et al. 2012Oberbeckmann S., Fuchs B.M., Meiners M. et al. 2012. Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea. Microb. Ecol. 63: 543-551.), but this was not the case for our area of study. Vibrios have a high plasticity in their genome and seem to be adaptable to changes in salinity (Cavallo and Stabili 2004Cavallo R.A., Stabili L. 2004. Culturable vibrios biodiversity in the Northern Ionian Sea (Italian coasts). Sci. Mar. 68: 23-29.). The significant positive correlations for % γ-proteobacteria and ammonia are also noteworthy. Ammonia has been adopted as a sewage water indicator since it is the result of urine decay. It is plausible that γ-proteobacteria, a group of generally fast-growing opportunistic bacteria, might have responded to these inputs of inorganic nitrogen or, alternatively, they might have been brought with the ammonia-rich terrestrial inputs.

In this study we have shown that vibrios may be associated with Ostreopsis blooms. As some Vibrio species are directly responsible for wound infections in marine waters (Ruppert et al. 2004Ruppert J., Panzig B., Guertler L. et al. 2004. Two cases of severe sepsis due to Vibrio vulnificus wound infection acquired in the Baltic Sea. Eur. J. Clin. Microbiol. Infect. Dis. 23: 912-915, Shets et al. 2006Shets F.M., van den Berg H.H., Demeulmeester A.A. et al. 2006. Vibrio alginolyticus infections in the Netherlands after swimming in the North Sea. Euro Surveill. 11: 3077., Reilly et al. 2011Reilly G.D., Reilly C.A., Smith E.G. et al. 2011. Vibrio alginolyticus-associated wound infection acquired in British waters, Guernsey. Euro Surveill., 16: 1994), it is plausible that part of the negative effects attributed to Ostreopsis blooms (e.g. Vila et al. 2012aVila M., Arin L., Battocchi C. et al. 2012a. Management of Ostreopsis blooms in recreational waters along the Catalan coast (NW Mediterranean Sea): cooperation between a research project and a monitoring program. Cryptogamie Algologie 33: 143-152.) might be related not only to palytoxin analogues but also to specific bacteria such us Vibrio sp.

CONCLUSIONTop

We have shown a positive relationship between the abundances of vibrios and of the benthic dinoflagellate Ostreopsis sp. at a coastal site of the NW Mediterranean. Abundance of bacteria of the genus Vibrio contributed up to 0.8% of the total planktonic bacterial community and up to 4.2% of the total epiphytic bacterial community, with a higher contribution in summer. Although most planktonic vibrios (mean of 83%) had a free-living lifestyle, particles in water constitute a niche for the Vibrio populations since they can occasionally represent up to 72% of the total bacterial community attached to particles.

ACKNOWLEDGEMENTSTop

This work was supported by the Spanish projects ICARO (200830I120), STORM (CTM2009-09352), DOREMI (CTM2012-34294), EBITOX (CTQ2008-06754-C04-04), OSTREORISK (CTM2014-53818-R) and ANIMA (CTM2015-65720), and through an FPI grant to E.B. of the Spanish Ministry of Economy and Competitiveness. We are very grateful to M. Mestre, C. Antequera and I. Forn for their assistance in the lab and we also thank the Aceña family of the Restaurant Pins Mar (Sant Andreu de Llavaneres), who kindly offered the use of their facilities in Llavaneres. Finally, we would like to thank Mariagrazia Giacobbe and an anonymous reviewer for their constructive comments on the manuscript.

REFERENCESTop

Alonso-Sáez L., Balagué V., Sà E.L., et al. 2007. Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH. FEMS. Microbiol. Ecol. 60: 98-112.
http://dx.doi.org/10.1111/j.1574-6941.2006.00276.x

Alonso-Sáez L., Vázquez-Domínguez E., Pinhassi J. et al. 2008. Factors controlling the year-round variability in carbon flux through bacteria in a coastal marine system. Ecosystems 11: 397-409.
http://dx.doi.org/10.1007/s10021-008-9129-0

Ashton M., Rosado W., Govind N.S. et al. 2003. Culturable and nonculturable bacterial symbionts in the toxic benthic dinoflagellate Ostreopsis lenticularis. Toxicon 42: 419-424.
http://dx.doi.org/10.1016/S0041-0101(03)00174-0

Asplund M.E., Rehnstam-Holm A.S., Atnur V., et al. 2011. Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area. Environ. Microbiol. 13: 2738-2751.

Baffone W., Tarsi R., Pane L. et al. 2006. Detection of free-living and plankton-bound vibrios in coastal waters of the Adriatic Sea (Italy) and study of their pathogenicity-associated properties. Environ. Microbiol. 8: 1299-1305.
http://dx.doi.org/10.1111/j.1462-2920.2006.01011.x

Barlaan E.A., Furukawa S., Kazuisha T. 2007. Detection of bacteria associated with harmful algal blooms from coastal and microcosm environments using electronic microarrays. Environ. Microbiol. 9: 690-702.
http://dx.doi.org/10.1111/j.1462-2920.2006.01188.x

Battocchi C., Totti C., Vila M., et al. 2010. Monitoring of toxic microalga Ostreopsis (Dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR based assay combined with light microscopy method. Mar. Pollut. Bull. 60:1074-1084.
http://dx.doi.org/10.1016/j.marpolbul.2010.01.017

Borrull E. 2011. Diversidad y actividad de la comunidad microbiana asociada a proliferaciones de fitobentos tóxico. Master thesis. Universitat de Barcelona.

Canigral I., Moreno Y., Alonso J.L., et al. 2010. Detection of Vibrio vulnificus in seafood, seawater and wastewater samples from a Mediterranean coastal area. Microbiol. Res. 165: 657-664.
http://dx.doi.org/10.1016/j.micres.2009.11.012

Cavallo R.A., Stabili L. 2004. Culturable vibrios biodiversity in the Northern Ionian Sea (Italian coasts). Sci. Mar. 68: 23-29.

De Paola A., Hopkins L.H., Peeler J.T., et al. 1990. Incidence of Vibrio parahaemolyticus in US coastal waters and oysters. Appl. Environ. Microbiol. 8: 2299-2302.

Doucette G.J., Kodama G., Franca S. 1998. Bacterial interactions with harmful algal bloom species: Bloom ecology, toxigenesis, and cytology. In: Andersen D.M., Cembella A.D., Hallegraeff G.M. (eds) The Physiological Ecology of Harmful Algal Blooms, NATO/ASI Series. Springer Verlag, Heidelberg, pp. 619-646.

Eiler A., Johansson M., Bertilsson S. 2006. Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas). Appl. Environ. Microbiol. 72: 6004-6011.
http://dx.doi.org/10.1128/AEM.00917-06

Froelich B., Ayrapetyan M., Oliver J.D. 2013. Integration of Vibrio vulnificus into marine aggregates and its subsequent uptake by Crassostrea virginica oysters. Appl. Environ. Microbiol. 79: 1454-1458.
http://dx.doi.org/10.1128/AEM.03095-12

Gallacher S., Flynn K.J., Franco J.M. et al. 1997. Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp (Dinophyta) in culture. Appl. Environ. Microbiol. 63: 239-245.

Grasshoff H., Ehrhardt M., Kremling K. 1983. Methods of Seawater Analysis. Verlag Chemie, Germany.

Groben R., Doucette G.J., Kopp M., et al. 2000. 16S rRNA targeted probes for the identification of bacterial strains isolated from cultures of the toxic dinoflagellate Alexandrium tamarense. Microb. Ecol. 39: 186-196.

Gonzalez D.J., Gonzalez R.A., Froelich B.A. et al. 2014. Non-native macroalga may increase concentrations of Vibrio bacteria on intertidal mudflats. Mar. Ecol. Prog. Ser. 505: 29-36.
http://dx.doi.org/10.3354/meps10771

Honsell G., Bonifacio A., De Bortoli M., et al. 2013. New insights on cytological and metabolic features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): A multidisciplinary approach. PLOS ONE, 8: e57291.
http://dx.doi.org/10.1371/journal.pone.0057291

Huggett M.J., Crocetti G.R., Kjelleberg S. 2008. Recruitment of the sea urchin Heliocidaris erythrogramma and the distribution and abundance of inducing bacteria in the field. Aquat. Microb. Ecol. 53: 161-171.
http://dx.doi.org/10.3354/ame01239

Kodama M., Doucette G.J., Green D.H. 2006. Relationships between bacteria and harmful algae. In: Granelli E. and Turner J.T. (eds) Ecology of Harmful Algae, Springer, pp: 243-255.
http://dx.doi.org/10.1007/978-3-540-32210-8_19

Lenes J.M., Walsh J.J., Barrow B.P. 2013. Simulating cell death in the termination of Karenia brevis blooms: implications for predicting aerosol toxicity vectors to humans. Mar. Ecol. Prog. Ser. 493: 71-81.
http://dx.doi.org/10.3354/meps10515

Lenoir S., Ten-Hage L., Turquet J. et al. 2004. First evidence of palytoxin analogues from an Ostreopsis mascarenensis (Dinophyceae) benthic bloom in Southwestern Indian Ocean. J. Phycol. 40: 1042-1051.
http://dx.doi.org/10.1111/j.1529-8817.2004.04016.x

Lopez-Joven C., de Blas I., Furones M.D. et al. 2015. Prevalences of pathogenic and non-pathogenic Vibrio parahaemolyticus in mollusks from the Spanish Mediterranean Coast. Front. Microbiol. 6: 736.
http://dx.doi.org/10.3389/fmicb.2015.00736

Lu Y. H., Chai T.J., Hwang D.F. 2000. Isolation of bacteria from toxic dinoflagellate Alexandrium minutum and their effects on algae toxicity. J. Nat. Toxins 9: 409-417.

Lyons M.M., Lau Y.T., Carden W.E. et al. 2007. Characteristics of marine aggregates in shallow-water ecosystems: Implications for disease ecology. Ecohealth 4: 406-420.
http://dx.doi.org/10.1007/s10393-007-0134-0

Main C.R., Salvitti L.R., Whereat E.B. et al. 2015. Community-level and species-specific associations between phytoplankton and particle-associated Vibrio species in Delaware’s inland bays. Appl. Environ. Microbiol. 81: 5703-5713.
http://dx.doi.org/10.1128/AEM.00580-15

Mangialajo L., Ganzin N., Accoroni S. et al. 2011. Trends in Ostreopsis proliferation along the Northern Mediterranean coasts. Toxicon 57: 408-420.
http://dx.doi.org/10.1016/j.toxicon.2010.11.019

Manz W., Amann R., Ludwig W., et al. 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria - problems and solutions. Syst. Appl. Microbiol.15: 593-600.
http://dx.doi.org/10.1016/S0723-2020(11)80121-9

Mizunoe Y., Wai S.N., Ishikawa T. et al. 2000. Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation. FEMS Microbiol. Lett. 186: 115-120.
http://dx.doi.org/10.1111/j.1574-6968.2000.tb09091.x

Mourino-Perez R.R., Worden A.Z., Azam F. 2003. Growth of Vibrio cholerae O1 in red tide waters off California. Appl. Environ. Microbiol. 69: 6923-6931.
http://dx.doi.org/10.1128/AEM.69.11.6923-6931.2003

Neogi S.B., Islam M.S., Nair G.B. et al. 2012. Occurrence and distribution of plankton-associated and free-living toxigenic Vibrio cholerae in a tropical estuary of a cholera endemic zone. Wet. Ecol. Manag. 20: 271-285.
http://dx.doi.org/10.1007/s11273-012-9247-5

Oberbeckmann S., Fuchs B.M., Meiners M. et al. 2012. Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea. Microb. Ecol. 63: 543-551.
http://dx.doi.org/10.1007/s00248-011-9990-9

Penna A., Vila M., Fraga S. et al. 2005. Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western Mediterranean Sea based on morphology, toxicity and internal transcribed spacer 5.8s rDNA sequences. J. Phycol. 41: 212-225.
http://dx.doi.org/10.1111/j.1529-8817.2005.04011.x

Pérez-Guzman L., Pérez-Matos A.E., Rosado W. et al. 2008. Bacteria associated with toxic clonal cultures of the dinoflagellate Ostreopsis lenticularis. Mar. Biotech. 10: 492-496.
http://dx.doi.org/10.1007/s10126-008-9088-7

Pernthaler A., Pernthaler J., Amann R. 2002. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68: 3094-3101.
http://dx.doi.org/10.1128/AEM.68.6.3094-3101.2002

Pinhassi J., Sala M.M., Havskum H. et al. 2004. Changes in bacterioplankton composition under different phytoplankton regimes. Appl. Environ. Microbiol. 70: 6753-6766.
http://dx.doi.org/10.1128/AEM.70.11.6753-6766.2004

Porter K.G., Feig F.Y. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948.
http://dx.doi.org/10.4319/lo.1980.25.5.0943

Reilly G.D., Reilly C.A., Smith E.G. et al. 2011. Vibrio alginolyticus-associated wound infection acquired in British waters, Guernsey. Euro Surveill., 16: 1994
http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19994

Ruiz-González C., Lefort T., Galí M. et al. 2012 Seasonal patterns in the sunlight sensitivity of bacterioplankton from Mediterranean surface coastal waters. FEMS Microbiol. Ecol. 79: 661-674
http://dx.doi.org/10.1111/j.1574-6941.2011.01247.x

Ruppert J., Panzig B., Guertler L. et al. 2004. Two cases of severe sepsis due to Vibrio vulnificus wound infection acquired in the Baltic Sea. Eur. J. Clin. Microbiol. Infect. Dis. 23: 912-915.
http://dx.doi.org/10.1007/s10096-004-1241-2

Sala M.M., Balagué V., Pedrós-Alio C. et al. 2005. Phylogenetic and functional diversity of bacterioplankton during Alexandrium spp. blooms. FEMS Microbiol. Ecol. 54: 257-267.
http://dx.doi.org/10.1016/j.femsec.2005.04.005

Shears N.T., Ross P.M. 2009. Blooms of benthic dinoflagellates of the genus Ostreopsis; an increasing and ecologically important phenomenon on temperate reefs in New Zealand and worldwide. Harmful Algae 8: 916-925
http://dx.doi.org/10.1016/j.hal.2009.05.003

Shets F.M., van den Berg H.H., Demeulmeester A.A. et al. 2006. Vibrio alginolyticus infections in the Netherlands after swimming in the North Sea. Euro Surveill. 11: 3077.

Simoni F., di Paolo C., Gori L. et al. 2004. Further investigation on blooms of Ostreopsis ovata, Coolia monotis, Prorocentrum lima on the macroalgae of artificial and natural reefs in the Northern Tyrrhenian Sea. Harmful Algae News 26: 5-7.

Szabo G., Preheim S.P., Kauffman K.M. et al. 2013. Reproducibility of Vibrionaceae population structure in coastal bacterioplankton. ISME J. 7: 509-519.
http://dx.doi.org/10.1038/ismej.2012.134

Takemura A.E., Chien D.M., Polz M.E. 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front. Microbiol. 5: 38.
http://dx.doi.org/10.3389/fmicb.2014.00038

Tamplin M.L., Gauzens M.L., Huq A. et al. 1990. Attachment of Vibrio-cholerae serogroup-O1 to zooplankton and phytoplankton of Bangladesh waters. Appl. Environ. Microb. 56: 1977-1980.

Thompson J.R., Randa M.A., Marcelino L.A. et al. 2004. Diversity and dynamics of a north Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70: 4103-4110.
http://dx.doi.org/10.1128/AEM.70.7.4103-4110.2004

Thompson J.R., Pacocha S., Pharino C. et al. 2005. Genotypic diversity within natural coastal bacterioplankton population. Science 307: 1311-1313.
http://dx.doi.org/10.1126/science.1106028

Tosteson T.R., Ballantine D.L., Tosteson C.G. et al. 1989. Associated bacterial-flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus-toxicus. Appl. Environ. Microbiol. 55: 137-141.

Tubaro A., Durando P., Del Favero G. et al. 2011. Case definitions for human poisonings postulated to palytoxins exposure. Toxicon 57: 478-495.
http://dx.doi.org/10.1016/j.toxicon.2011.01.005

Vezzulli L., Previati M., Pruzzo C. et al. 2010. Vibrio infections triggering mass mortality events in a warming Mediterranean Sea. Environ. Microbiol. 12: 2007-2019.
http://dx.doi.org/10.1111/j.1462-2920.2010.02209.x

Vezzulli L., Brettar I., Pezzati E. et al. 2012. Long-term effects of ocean warming on the prokaryotic community: evidence from the vibrios. ISME J. 6: 21-30.
http://dx.doi.org/10.1038/ismej.2011.89

Vezzulli, L., Colwell R.R., Pruzzo C. 2013. Ocean warming and spread of pathogenic vibrios in the aquatic environment. Microb. Ecol. 65: 817-825.
http://dx.doi.org/10.1007/s00248-012-0163-2

Vila M, Garcés E., Masó M. 2001. Potentially toxic epiphytic dinoflagellate assemblages on macroalgae in the NW Mediterranean. Aquat. Microb. Ecol. 26: 51-60.
http://dx.doi.org/10.3354/ame026051

Vila M., Masó M., Sampedro N. et al. 2008. The genus Ostreopsis in recreational waters of the Catalan Coast and Balearic Islands (NW Mediterranean Sea): is this the origin of human respiratory difficulties? In: Proceedings of the 12 International Conference on Harmful Algae, pp. 334-336.

Vila M., Arin L., Battocchi C. et al. 2012a. Management of Ostreopsis blooms in recreational waters along the Catalan coast (NW Mediterranean Sea): cooperation between a research project and a monitoring program. Cryptogamie Algologie 33: 143-152.
http://dx.doi.org/10.7872/crya.v33.iss2.2011.143

Vila M., Riobó P., Bravo I. et al. 2012b. A three-year time series of toxic Ostreopsis blooming in a NW Mediterranean coastal site: Preliminary results. In: Pagou P. and Hallegraeff G. (eds). Proceeding of the 14th International Conference on Harmful Algae. ISSHA and IOC of UNESCO, pp. 111-113.

Vila M., Abós-Herràndiz R., Isern-Fontanet J., et al. 2016. Establishing the link between Ostreopsis cf. ovata blooms and human health impacts using ecology and epidemiology. Sci. Mar. 80S1: 107-115.
http://dx.doi.org/10.3989/scimar.04395.08A

Yentsch C.S., Menzel D.W. 1963. A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res. 10: 221-231.
http://dx.doi.org/10.1016/0011-7471(63)90358-9

Worden A.Z., Seidel M., Smriga S. et al. 2006. Trophic regulation of Vibrio cholerae in coastal marine waters. Environ. Microbiol. 8: 21-29.
http://dx.doi.org/10.1111/j.1462-2920.2005.00863.x



Copyright (c) 2016 Consejo Superior de Investigaciones Científicas (CSIC)

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.


Contact us scimar@icm.csic.es

Technical support soporte.tecnico.revistas@csic.es